In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

Abstract

To what extent, how and when axons respond to injury in the highly interconnected grey matter is poorly understood. Here we use two-photon imaging and focused ion beamscanning electron microscopy to explore, at synaptic resolution, the regrowth capacity of several neuronal populations in the intact brain. Time-lapse analysis of 4100 individually ablated axons for periods of up to a year reveals a surprising inability to regenerate even in a glial scar-free environment. However, depending on cell type some axons spontaneously extend for distances unseen in the unlesioned adult cortex and at maximum speeds comparable to peripheral nerve regeneration. Regrowth follows a distinct pattern from developmental axon growth. Remarkably, although never reconnecting to the original targets, axons consistently form new boutons at comparable prelesion synaptic densities, implying the existence of intrinsic homeostatic programmes, which regulate synaptic numbers on regenerating axons. Our results may help guide future clinical investigations to promote functional axon regeneration.

Synaptic Elimination and Protection after Minimal Injury Depend on Cell Type and Their Prelesion Structural Dynamics in the Adult Cerebral Cortex

“…We defined axons as attempting to regrow (regrowing axons) if the elongation from the cut stump reached the lesion site and exceeded twice the maximum noise measurement (i.e., Ͼ6 m; the maximum difference in measurements between fiducial points over repeated imaging sessions is 3 m; De Paola et al, 2006;Canty et al, 2013).…”

“…More than one-half of lesioned axons (48 axons in the adult brain; 12 axons in the juvenile brain) were observed chronically for Ͼ2 weeks and up to 1 year postlesion. Identified axons were severed by inducing a laser lesion with a single circular spot scan (a binary mask of 4 m diameter, ϳ600 mW at the back focal plane (i.e., 1 order of magnitude higher than the power used for imaging), 800 nm and 30 s dwell time; Canty et al, 2013). We quantified the diameter of the first 10 lesions based on the fluorescent mark left and found very low variability (see Results for details).…”

“…We measured the length of the surviving axon, relative to the lesion site, at a variety of intervals postlesion for both regrowing (n ϭ 17 distinct axons in total across all time points) and nonregrowing axons (n ϭ 66 distinct axons in total across all time points). Regrowing and nonregrowing axons represent two fundamentally different axonal populations in the adult brain (Canty et al, 2013).…”

“…Axons were imaged individually through a cranial window and classified according to their morphology based on previous anatomical reconstructions and findings (De Paola et al, 2006;see Materials and Methods). To study synaptic reorganization over time scales including both immediate and more delayed responses to injury we used twophoton laser-mediated axotomy, which uniquely allows paired, localized lesion is not associated with a prominent glial scar and infiltrating macrophages (Ylera et al, 2009;Canty et al, 2013), allowing to tease apart intrinsic versus extrinsic determinants of axon and synaptic reorganization. We were able to transect axons within defined lengths away from the distal end in most neurons (for 58 of 83 axons, mean portion cut ϭ 542 Ϯ 38 m, range ϳ150 -1289 m).…”

“…Animals were killed at defined time points postlesion (11 d, 1 m, 3 m, 6 m, and 12 m) or where the health of the mouse became compromised (maximum age postlesion 12 months). During one of the imaging sessions, overlapping low-magnification overviews of the labeled neuropil were collected to help identification of axons in the fixed brain (Canty et al, 2013).…”